Single-Molecule PCR on Microparticles in Water-In-Oil Emulsions

ABSTRACT

Modulation of the viscosity of the oil phase of a microemulsion used for amplification of DNA on a bead increases the homogeneity of product beads and the amount of amplified DNA per bead. Moreover the number of separate microemulsion populations that can be formed in parallel is increased using multi-well plates and mixer mill disrupter machines designed to lyse biological samples.

The entire disclosure of U.S. provisional application Ser. No.60/814,585 filed Jun. 19, 2006, is expressly incorporated herein.

The U.S. government retains certain rights in the invention by virtue ofgrants from the NIH including CA 43460, CA57345, and CA 62924.

TECHNICAL FIELD OF THE INVENTION

This invention is related to the area of DNA analysis. In particular, itrelates to the amplification and segregation of single species of DNAmolecules.

BACKGROUND OF THE INVENTION

The most important biotechnological advances made in the 20^(th) centuryinvolved methods that convert a single DNA molecule into a population ofidentical DNA molecules. The first wave of techniques for this purposeemployed cells (cloning) and the second wave employed PCR. Cloning wasadvantageous in that the populations emanating from individual moleculeswere inherently separated through this process. In contrast, PCR-basedmethods required individual compartments (tubes) for each template ifthe products were to be kept separate. Emulsion PCR overcame thisdisadvantage by miniaturizing the compartments so that millions oftemplates could be individually amplified within a single tube.

BEAMing (beads, emulsions, amplification, and magnetics) built onemulsion PCR by keeping products formed within each compartment togetheronce the emulsions were broken. This was accomplished through (i)inclusion of beads within the compartments and (ii) ensuring that onestrand of the PCR product is bound to the beads. After amplification,each bead is coated with thousands of copies of the single DNA moleculepresent in the compartment that contained that bead and these beadscould easily be recovered with a magnet or by centrifugation.

Beads obtained via BEAMing accurately reflect the DNA diversity presentin the template population and can be used to determine what fraction ofa DNA population contains a specific mutation. Because each beadcontains thousands of molecules of the identical sequence, the signal tonoise ratio obtained with hybridization or enzymatic assays is extremelyhigh. Millions of beads can be analyzed within minutes usingconventional flow cytometry or optical scanning instruments. The DNAbound to the beads also provides excellent templates for high-throughputsequencing.

There is a continuing need in the art to improve the throughput of DNAamplification to improve analysis of DNA and genetic diagnoses.

SUMMARY OF THE INVENTION

According to one embodiment of the invention a method for analyzingnucleotide sequence variations is provided. Microemulsions comprising anoil phase and an aqueous phase are formed. The aqueous phase comprisesone or more species of analyte DNA molecules. From 10-30% (v/v) of themicroemulsions is the aqueous phase and from 70-90% (v/v) of themicroemulsions is the oil phase. The oil phase comprises one or more lowviscosity hydrocarbons with a viscosity less than 20 mPas at 25° C. inan amount from 60-85% (v/v) of the oil phase, one or more high viscosityhydrocarbons having a viscosity of greater than 20 mPas at 25° C. in anamount from 10-30% (v/v), and an emulsifier in an amount from 5-10%(v/v). Analyte DNA molecules in the microemulsions are amplified in thepresence of reagent beads. The reagent beads are bound to a plurality ofmolecules of a primer for amplifying the analyte DNA molecules. Productbeads are formed which are bound to a plurality of copies of one speciesof analyte DNA molecule. The product beads are separated from analyteDNA molecules which are not bound to product beads. A sequence featureof the one species of analyte DNA molecule which is bound to the productbeads is determined.

A liquid composition is provided by the present invention. It comprisesa plurality of microemulsions forming aqueous compartments wherein atleast a portion of said aqueous compartments comprise a bead, apolynucleotide template, and oligonucleotide primers for amplifying saidtemplate. At least a portion of the oligonucleotide primers is bound tothe bead. The microemulsions comprise an oil phase and an aqueous phase.From 10-30% (v/v) of the microemulsions is the aqueous phase and from70-90% (v/v) of the microemulsions is the oil phase. The oil phasecomprises one or more low viscosity hydrocarbons with a viscosity lessthan 20 mPas at 25° C. in an amount from 60-85% (v/v) of the oil phase,one or more high viscosity hydrocarbons having a viscosity of greaterthan 20 mPas at 25° C. in an amount from 10-30% (v/v), and an emulsifierin an amount from 5-10% (v/v).

Another embodiment of the invention is a method for isolating nucleotidesequence variants. Microemulsions comprising an oil phase and an aqueousphase are formed. The aqueous phase comprises one or more species ofanalyte DNA molecules. From 10-30% (v/v) of the microemulsions is theaqueous phase and from 70-90% (v/v) of the microemulsions is the oilphase. The oil phase comprises one or more low viscosity hydrocarbonswith a viscosity less than 20 mPas at 25° C. in an amount from 60-85%(v/v) of the oil phase, one or more high viscosity hydrocarbons having aviscosity of greater than 20 mPas at 25° C. in an amount from 10-30%(v/v), and an emulsifier in an amount from 5-10% (v/v). Analyte DNAmolecules in the microemulsions are amplified in the presence of reagentbeads. The reagent beads are bound to a plurality of molecules of aprimer for amplifying the analyte DNA molecules. Product beads areformed which are bound to a plurality of copies of one species ofanalyte DNA molecule. The product beads are separated from analyte DNAmolecules which are not bound to product beads. Product beads which arebound to a plurality of copies of a first species of analyte DNAmolecule are isolated from product beads which are bound to a pluralityof copies of a second species of analyte DNA molecule.

Another aspect of the invention is an improvement of a method foranalyzing nucleotide sequence variations in which microemulsionscomprising one or more species of analyte DNA molecules are formed, theanalyte DNA molecules in the microemulsions are amplified in thepresence of reagent beads, wherein the reagent beads are bound to aplurality of molecules of a primer for amplifying the analyte DNAmolecules, product beads are formed which are bound to a plurality ofcopies of one species of analyte DNA molecule, the product beads areseparated from analyte DNA molecules which are not bound to productbeads, and a sequence feature of the one species of analyte DNA moleculewhich is bound to the product beads is determined. The improvement isthe use of a tissue mixer mill disrupter to form a plurality of separatemicroemulsion populations simultaneously in a multi-well plate using anda metal ball in each well.

These and other embodiments which will be apparent to those of skill inthe art upon reading the specification provide the art with tools formore efficiently analyzing DNA variations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 diagrams the BEAMing procedure. References to the steps in theprotocol are indicated along with each key step.

FIG. 2 shows a phase contrast micrograph at 400× of emulsions depositedin the well of a 48-well tissue culture plate. For reference, the beads(arrows) are 1.05 microns in diameter.

FIGS. 3A-3B show an analysis of bead populations with flow cytometry.Circled population in FIG. 3A (single beads) is shown in FIG. 3B,representing beads labeled with a sequence-specific FITC signal.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have developed methods which improve on former methods forpracticing BEAMing. The methods described below provide for morehomogenous populations of beads that have a higher amount of DNA on eachbead at the end of cycling. Moreover, the use of the tissue lyser andmetal beads to make a plurality of microemulsions in parallel massivelyincrease throughput from single or double parallel samples to hundredsof parallel samples.

The aqueous phase in the microemulsions typically comprises at least 10,15, 20, or 25, and up to 30% (v/v) of the microemulsions. The oil phasetypically comprises at least 70, 75, 80, or 85, and up to 90% (v/v) ofmicroemulsions. The emulsifier, although amphiphilic, is considered partof the oil phase. It is believed to align at the interface of the twophases upon formation of an emulsion. Hydrocarbons which form the oilphase are typically oils or waxes. These are characterized by theirviscosities as shown below:

Very low <5 mPas at 25° C. Low 5-10 mPas at 25° C. Medium 10-20 mPas at25° C. High 20-50 mPas at 25° C. Very high >50 mPas at 25° C.

A lower viscosity oil (defined as a very low, low, and/or medium) iseasier to work with than a high viscosity oil. The low viscosity oil canhave a viscosity of less than 10 or less than 5 mPas at 25° C. However,mixing a small amount of the higher viscosity oil into the oil phaseprovides increased uniformity among product beads and higheramplification levels. The high viscosity oil can be within a range of10-30, 20-25, or 22-26 mPas at 25° C. The majority of the oil phase canbe eithera very low, a low, a medium, or a mixture of such oils orwaxes. The proportion of lower viscosity oil to high viscosity oil canvary from 85:10 to 60:30. At least 60, 65, 70, 75, or 80 and up to 85%(v/v) of the oil phase can be a lower viscosity oil. At least 10, 15,17, 20, or 25, and up to 23, 25, 27, or 30% (v/v) of the oil phase canbe a high viscosity oil. The emulsifier can comprise at least 5, 6, 7,8, or 9% and up to 10% (v/v) of the oil phase.

After amplification, product beads can be analyzed to determine asequence feature of the DNA bound to them. Any method for determining asequence feature can be used, including, hybridization, primerextension, and nucleotide sequencing. Alternatively, product beads canbe analyzed by FACs analysis, i.e., a technique for separating ordistinguishing two different species of nucleotide molecule. Other meansof analysis can be used as is convenient.

Beads according to the present invention are also known as microspheresor microparticles. Particle sizes can vary between about 0.1 and 10microns in diameter. Typically beads are made of a polymeric material,such as polystyrene, although nonpolymeric materials such as silica canalso be used. Other materials which can be used include styrenecopolymers, methyl methacrylate, functionalized polystyrene, glass,silicon, and carboxylate. Optionally the particles aresuperparamagnetic, which facilitates their purification after being usedin reactions.

Beads can be modified by covalent or non-covalent interactions withother materials, either to alter gross surface properties, such ashydrophobicity or hydrophilicity, or to attach molecules that impartbinding specificity. Such molecules include without limitation,antibodies, ligands, members of a specific-binding protein pair,receptors, nucleic acids. Specific-binding protein pairs includeavidin-biotin, streptavidin-biotin, and Factor VII-Tissue Factor.

Beads, after being prepared according to the present invention asproduct beads, have more than one copy of the same nucleic acid moleculebound to them. Preferably each bead is bound to at least 10, 50, 100,500, or 1000 molecules of the same nucleic acid sequence. In somecircumstances some of the product beads are bound to more than one typeof nucleic acid molecule. These product beads are generally less usefulin the analysis of ratios of genetic sequences in a population ofgenetic sequences. Such product beads can be readily discriminated andso will not distort the analysis.

A population of product beads will often comprise two or more types ofnucleic acids. Such a population is heterogeneous with respect to thenucleic acids. Desirably, a substantial proportion of the product beadscomprise only one type of nucleic acid per bead. A substantialproportion can be for example, at least 1%, at least 5%, at least 10%,or at least 50%. A product bead with only one type of nucleic acid perbead is termed homogeneous. Homogeneous beads with only one type ofnucleic acid per bead include those with nucleic acids containing errorsdue to errors in polymerase chain reaction. A product bead with twotypes of nucleic acid per bead is termed heterogeneous. Although notwishing to be bound by any particular theory, heterogeneous productbeads are thought to result from aqueous compartments which have morethan two molecules of template of non-identical sequence. A populationof product beads can be heterogeneous as a population but containindividual product beads that are homogeneous

Individual product beads preferably comprise more than one copy oftemplate analyte molecule. Each bead may comprise at least 10, at least50, at least 100, at least 500, or at least 1000 copies of templateanalyte. If the bead is homogeneous, each of those copies will beidentical.

Populations of product beads can be maintained in a liquid suspension.Alternatively they can be sedimented and dried or frozen. The latteralternatives may be beneficial for storage stability.

Analysis of populations of product beads can be useful fordistinguishing between many kinds of genetic variants. Polynucleotidescan be distinguished which differ by as little as a single nucleotidepolymorphism (SNP), by the presence or absence of a mutation, by thepresence or absence of an insertion or deletion, by the presence orabsence of a non-single nucleotide polymorphism. Thus populations ofproduct beads may be heterogeneous with regard to these geneticvariations.

One very convenient way for distinguishing genetic variants, i.e.,determining a sequence feature of the analyte, is by differentiallylabeling the variants with fluorescent dyes. Such labeling can beaccomplished by hybridization of a fluorescently labeled oligonucleotideprobe to one species of polynucleotide. Alternatively, a fluorescentlylabeled antibody can be used to specifically attach to oneoligonucleotide probe that hybridizes to a particular genetic variant.Such antibody binding can be, for example, mediated by a protein orpolypeptide which is attached to an oligonucleotide hybridization probe.Of course, other means of labeling polynucleotides as are known in theart can be used without limitation. Another means of labeling differentpolynucleotide species is by primer extension. Primers can be extendedusing labeled deoxyribonucleotides, such as fluorescently labeleddeoxyribonucleotides.

Populations of product beads can be used as templates. Template analytemolecules on the product beads can be analyzed to assess DNA sequencevariations by hybridization, primer-extension methods, massspectroscopy, and other methods commonly used in the art. Templateanalyte molecules on product beads can be employed for solid phasesequencing. In one solid phase sequencing technique, product beads arearrayed by placing them on slides spotted with complementaryoligonucleotides. In another solid phase sequencing technique, productbeads are placed into individual wells. In still another solid phasesequencing technique product beads are incorporated into acrylamidematrices (with or without subsequent polony formation). Sequencingreactions can be performed with any solid phase sequencing method, suchas those using unlabeled nucleotide precursors (e.g., pyrosequencing, asdescribed in Ronaghi et al., Anal. Biochem. 267: 65-71, 1999) or labelednucleotides (e.g., photocleavable reagents described by Mitra et al.,Anal. Biochem. 320:55-65, 2003). Product beads can thus be used for andfacilitate multiple parallel sequencing. Product beads can also be usedin sequencing employing Type IIS restriction endonucleases. Productbeads can also be used to provide templates for conventionaldideoxynucleotide sequencing. To obtain useful data upon sequenceanalysis, a homogeneous template population is desirable. To provide ahomogenous template population, product beads can be diluted, separated,or otherwise isolated so that each sequencing reaction contains a singleproduct bead. Alternatively, product beads can be sorted to providepopulations of beads with a single species of template.

Oligonucleotide primers can be bound to beads by any means known in theart. They can be bound covalently or non-covalently. They can be boundvia an intermediary, such as via a protein-protein interaction, such asan antibody-antigen interaction or a biotin-avidin interaction. Otherspecific binding pairs as are known in the art can be used as well. Toachieve optimum amplification, primers bound to the bead may be longerthan necessary in a homogeneous, liquid phase reaction. Oligonucleotideprimers may be at least 12, at least 15, at least 18, at least 25, atleast 35, or at least 45 nucleotides in length. The length of theoligonucleotide primers which are bound to the beads need not beidentical to that of the primers that are in the liquid phase. Primerscan be used in any type of amplification reaction known in the art,including without limitation, polymerase chain reaction, isothermalamplification, rolling circle amplification, self-sustaining sequencereplication (3SR), nucleic acid sequence-based amplification (NASBA),transcription-mediated amplification (TMA), strand-displacementamplification (SDA), and ligase chain reaction (LCR).

Microemulsions are made by stirring or agitation of oil, aqueous phase,and detergent. The microemulsions form small aqueous compartments whichhave an average diameter of 0.5 to 50 microns. The compartments may befrom 1 to 10 microns, inclusive, from 11 to 100 microns, inclusive, orabout 5 microns, on average. All such compartments need not comprise abead. Desirably, at least one in 10,000 of said aqueous compartmentscomprise a bead. Typically from 1/100 to 1/1 or from 1/50 to 1/1 of saidaqueous compartments comprise a bead. In order to maximize theproportion of beads which are homogeneous with respect tooligonucleotide, it is desirable that on average, each aqueouscompartment contains less than 1 template molecule. Aqueous compartmentswill also desirably contain whatever reagents and enzymes are necessaryto carry out amplification. For example, for polymerase chain reaction(PCR) the compartments will desirably contain a DNA polymerase anddeoxyribonucleotides. For rolling circle amplification a DNA polymeraseand a generic DNA circle may be present.

Emulsions can be “broken” or disrupted by any means known in the art.One particularly simple way to break the emulsions is to add moredetergent. Detergents which can be used include, but are not limited toTriton X100, Laureth 4, Nonidet.

Sample DNA for amplification and analysis according to the presentinvention can be genomic DNA, cDNA, PCR products of genomic DNA, or PCRproducts of cDNA, for example. Samples can be derived from a singleindividual, for example, from a body sample such as urine, blood,sputum, stool, tissue or saliva. Samples can also be derived from apopulation of individuals. The individuals can be humans, but can be anyorganism, plant or animal, eukaryotic or prokaryotic, viral ornon-viral.

Any type of probe can be used for specific hybridization to theamplified polynucleotides which are bound to the beads. Fluorescentlylabeled probes are useful because their analysis can be automated andcan achieve high throughput. Fluorescence activated cell sorting (FACS)permits both the analysis and the isolation of different populations ofbeads. One type of fluorescently labeled probe that can be used is amodified molecular beacon probe. These probes have stem-loop structuresand an attached fluorescent moiety on the probe, typically on one end ofthe probe, sometimes attached through a linker. Unlike standardmolecular beacon probes, modified molecular beacon probes do not have aquenching moiety. The modified molecular beacon probe can have thefluorescent moiety attached on either end of the probe, 5′ or 3′. Onesuch probe will hybridize better to a wild-type sequence than to amutant. Another such probe will hybridize better to a mutant sequencethan to the wild type. Still other probes will preferably hybridize toone polymorphic variant over another.

High fidelity DNA polymerases which can be used are those which providea higher rate of fidelity (lower rate of errors) than Taq polymerase.Preferably these provide an error rate of less than 10⁻⁵, morepreferably an error rate of less than 5×10⁻⁶, and even more preferablyan error rate of less than 10⁻⁶. Suitable polymerases include: Phusion™DNA polymerase (NEB), Taq High Fidelity™, and PfuUltra™. These are usedin a thermal cycling polymerase chain reaction, as is conventional inthe art.

Microemulsions are formed with beads and primers as previously taught.Because BEAMing requires thermal cycling, an emulsifier which isthermostable can be used. One such emulsifier is Abil® EM90(Degussa-Goldschmidt Chemical, Hopewell, Va.). Other such emulsifierscan be used as are known in the art. Increased molecular weight ofemulsifiers correlates with increased thermostability.

Amplicons can be any size which is efficiently amplified usingpolymerase chain reaction. In the case of templates obtained from serumof cancer patients, amplicons are preferably shorter than or equal to300 bp, or shorter than or equal to 200 bp, or shorter than or equal to100 bp. Templates from serum of colon cancer patients are apparentlydegraded to small sizes. Thus amplification of a smaller ampliconresults in a more efficient and sensitive detection. The dependence ofdetection on size is quite strong as shown in FIG. 1.

Single base extension reaction with differentially labeleddideoxynucleotides provides a sensitive means for detecting sequencefeatures. If upon detection of products, individual beads are found withmultiple, distinct labels, for example, representing a mutant and a wildtype nucleotide, they can be discarded from further analysis. Multiple,distinct labels in this context indicates that a bead was present in amicroemulsion with two distinct templates of analyte DNA, rather thanthe desired single template, or that an error occurred early in anamplification reaction in a microemulsion, such that the erroneous andthe correct templates were both amplified.

One means for detecting a sequence feature on an amplicon bound to abead employs a single base extension (SBE) reaction. This reactiontypically employs labeled dideoxynucleotide triphosphates to ensure thatonly a single monomer addition occurs. Dideoxynucleotide triphosphatescan be conveniently labeled with any type of detectable label, includingradioactive, fluorescent, and luminescent moieties. Different labels canbe attached to different dideoxynucleotide triphosphates (ddNTPs) sothat different products can be detected in the same sample. Prior toaddition of all reagents necessary for initiation of the SBE reaction,unlabeled ddNTPs can be added to block non-specific extension. Typicallyat least one unlabeled ddNTP is added at a concentration five to 40 foldhigher than the concentration of the labeled ddNTPs. Preferably theconcentration is at least ten to twenty times higher. For example, if Ais the mutant base and C is the wild-type base, during the SBE, we canuse Rox-ddATP for the mutant, FITC-ddCTP for the wild type, ddGTP andddTTP for blocking the nonspecific extension at the ratio of1:2-10:20:20. The unlabeled ddNTPs reduce nonspecific incorporation.

Another optional step for improving the specificity and/or sensitivityof the SBE reaction is to denature the double stranded nucleic acidduplexes attached to the beads prior to the SBE reaction. For example,the double strands can be heated or treated with sodium hydroxide. Afterthe separation of the two strands, the single strands which are notbound to the beads can be separated from the beads and the bead-boundstrands, and the single strands can be discarded.

If desired, yet another step of amplification can be used after themicroemulsions are broken. This step typically employs isothermalamplification, also known as rolling circle amplification. In order togenerate the rolling circle, a molecular inversion probe or a padlockprobe can be used. They probe may require filling-in, or not, prior to atemplate-driven ligation reaction to generate a circle. If filling-in isrequired the region to be filled in will typically be from 1 to 30nucleotides. The isothermal amplification can amplify the ultimatelydetected signal quite significantly. After isothermal amplification, asequence feature can be detected using SBE (single base extension)reaction, as described above. Alternatively, the nucleotide sequence ofthe amplicon on the beads can be determined by any sequencing methodknown in the art, including sequencing-by-synthesis.

Samples which may be used as sources of analyte DNA include blood,plasma, urine, stool, sputum, tears, saliva, and bone marrow. Solidtissues can also provide analyte DNA. Samples can be obtained fromcancer patients, from related family members, from pregnant women, andfrom neonates. Sources of analyte DNA may be treated, for example withtest agents, and the effects of the test agents on the analyte DNA canbe determined.

While this protocol is an advancement of the current amplificationmethods, there are some limitations to the technology. One limitation isthe size of the PCR products that can be polymerized on the beads.Though we have successfully amplified sequences as long as 2,700 bp viaBEAMing, the yield of products of this large size is less than 5% ofthat achieved with amplicons <110 bp. Steric hindrance at the beadsurface is probably responsible for the lower efficiency with longamplicons. This could conceivably be overcome in the future by usingdifferent beads with more enzyme-compatible surfaces or differentpolymerases (though no commercially available polymerases have so farbeen more efficient than the one described above).

Another limitation of the method is the need to prepare a differentemulsion for each amplicon to be queried. This limitation has beenovercome in part by the new emulsion-making procedure described herein,which can in principle be automated and allow simultaneous generation of192 emulsions. It is possible that multi-parallel microfluidics could beused to generate greater numbers of emulsions, using processes such asthose recently described′. Microfluidics also has the capacity togenerate more homogeneous emulsions than methods based on shear forcesuch as the one described herein.

There are other technologies that can achieve compartmentalized PCRusing single molecules as templates. These include amplifications in thewells of picotiter plates⁶ or in polonies³. These methods do not yieldas many compartments as BEAMing but are adequate for severalapplications. BEAMing also offers the advantage that little specialequipment, for either preparation of emulsions or their analysis, isrequired other than what is routinely available at most institutions.

Mastrobattista et al. have recently described a method to produce singletemplate amplifications in water-oil-water (w/o/w) emulsions that can bedirectly used for flow cytometry⁷. Though the purposes for devising thismethod were distinct from the diagnostic applications driving thedevelopment of BEAMing, the droplets formed in the w/o/w-based procedurecould be used for variant detection if a fluorescent probe, such as aMolecular Beacon, were incorporated into the aqueous phase. This wouldallow querying one or a few variations within an amplicon, while BEAMingallows query of any variant within an amplicon.

There have been several recent publications that described applicationsof BEAMing. In addition to its use as templates for high-throughputsequencing^(3,8), it has been employed for the quantification ofmutations in plasma samples of cancer patients⁴, the directdetermination of polymerase error rates and the identification oftranscription factor targets⁹.

The above disclosure generally describes the present invention. Allreferences disclosed herein are expressly incorporated by reference. Amore complete understanding can be obtained by reference to thefollowing specific examples which are provided herein for purposes ofillustration only, and are not intended to limit the scope of theinvention.

Example 1 Materials

Reagents

Binding buffer: 5 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 1 M NaClBreaking buffer: 10 mM Tris-HCl (pH 7.5), 1% Triton-X100, 1% SDS, 100 mMNaCl, 1 mM EDTA48-well cell culture plate (Corning, 3548)Deoxynucleotide triphosphates (dNTPs) mix (10 mM each; USB, 77212)

FACS Sheath Solution (BD Biosciences, 342003)

5× hybridization buffer: 75 mM Tris-HCl (pH 9.5), 33.5 mM MgCl₂, 25%formamide CautionMicroamp clear adhesive film (Applied Biosystems, 4306311)1.5 ml microcentrifuge tubes (Screw cap, Corning, 430909)

Mineral oil (Sigma, M3516)

MyOne streptavidin-coated magnetic beads, hydrophilic, 1-μm diameter (10mg/ml; 7-12×10⁹ beads/ml; Invitrogen, 650-01)

0.1 M NaOH Caution

10× PCR buffer: 670 mM Tris-HCl (pH 8.8), 166 mM (NH₄)₂SO₄, 100 mM(3-mercaptoethanol,

11.7 mM MgCl₂ Caution

Oil/emulsifier mix: 7% (w/v) ABIL WE09 in Tegosoft DEC (DegussaGoldschmidt Chemical; available from authors as it is only sold inamounts that are much larger than needed for laboratory experiments).Store mixture at room temperature for no longer than two days.96-well PCR plates (Denville Scientific, C18096X)Phusion/iProof high-fidelity DNA polymerase, (2 U/μl; NEB/Biorad,F-530L/172-5302)Platinum Taq DNA polymerase (5 U/μl; Invitrogen, 10966-034)Quant-iT PicoGreen dsDNA assay kit (Invitrogen, P-7589)Stainless steel beads (5 mm; Qiagen, 69982)96-well storage plates (1.2 ml; round well; round bottom; Abgene,AB-0564)TE buffer: 10 mM Tris-HCl (pH 7.5), 1 mM EDTATK buffer: 20 mM Tris-HCl (pH 8.4), 50 mM KCl

Oligonucleotides

Six oligonucleotide primers are required for a single BEAMingexperiment, as illustrated in FIG. 1. Primers 1 to 3 are gene-specificwhile primers 4 to 6 are universal. We generally obtain oligonucleotidesfrom IDT.

Primer3 (domain name:frodo.wi.mit.edu/cgi-bin/primer3, document:primer3_www.cgi) is used to design the gene-specific portions of theprimers. These sequences should have a T_(m) of ˜60° C. and be 18 to 27nucleotides in length.

Primers 1 and 2 are used for pre-amplification of the DNA template.Primer 1 contains a universal sequence (Tag 1,5′-TCCCGCGAAATTAATACGAC-3; SEQ ID NO: 1) on its 5′-end and a sequencehomologous to the gene of interest at its 3′ end. Primer 2 contains asecond universal sequence (Tag 2, 5′-GCTGGAGCTCTGCAGCTA-3; SEQ ID NO: 2)on its 5′-end and a sequence homologous to the gene of interest at its3′ end.

Primer 3 is used for detection of amplification products on beads and islabeled with a fluorescent group (e.g., FAM) at its 5′ end.

Primer 4 is bound to the beads. It is doubly biotinylated at its 5′ endand has a PEG 18 spacer and a thymidine base between the biotins and theTag1 sequence, e.g., 5′-Dual biotin-Spacer18-T-TCCCGCGAAATTAATACGAC-3′;SEQ ID NO: 3.

Primers 5 and 6 are unmodified oligonucleotides with the Tag1 and Tag2sequences, respectively.

The dual biotin group is essential to keep the oligonucleotide attachedto the streptavidin-coated beads during thermal cycling′.

Equipment

Centrifuge with swinging buckets for microtiter plates (4K15;Qiagen/Sigma)Compression pads for a thermal cycler (Thermo Hybaid)Flow cytometer (LSRII, BD Biosciences)MPC-S and MPC-9600 magnetic separators (Invitrogen, 120-20D and 120-06D)

ND-1000 Spectrophotometer (NanoDrop Technologies)

TissueLyser mixer mill with adaptor plates from the 2× 24 adaptor set(Qiagen, 85210 and 69982)Single-bead dispenser (Qiagen, 69965)

Example 2 Procedure

Note that all steps are performed at room temperature except whereindicated otherwise.

Pre-Amplification of DNA Samples

-   1. Set up a 50 μl PCR reaction for the initial amplification of the    target region, as follows:

Primer 1 (10 μM) 1 μl Primer 2 (10 μM) 1 μl Template DNA (in water) 15μl  dNTPs mix 1 μl 5x Phusion HF buffer 10 μl  Water 21.5 μl   PhusionDNA polymerase (2 U/μl) 0.5 μl  

-   -   Add the components in the order listed. Overlay PCR reaction        with 15 μl mineral oil to prevent evaporation during the        temperature cycling. Other DNA polymerases can also be used for        this pre-amplification, depending on the fidelity required for        the specific application².    -   If the template DNA is complex, such as mammalian cellular DNA,        then we generally use 3 to 30 ng per PCR (1000-10,000 haploid        genome equivalents).

-   2. Place the reaction in a thermal cycler and amplify the DNA    fragment according to the following touchdown program:

Cycle number Denaturation Annealing Polymerization 1  1 min at 98° C.2-4 10 s at 98° C. 10 s at 70 s 10 s at 72° C. 5-7 10 s at 98° C. 10 sat 67 s 10 s at 72° C.  8-10 10 s at 98° C. 10 s at 64 s 10 s at 72° C.11-40 10 s at 98° C. 10 s at 61 s 10 s at 72° C.

-   -   It is possible to reduce the number of PCR cycles to minimize        polymerase induced sequence errors. Touchdown PCR conditions        minimize the formation of nonspecific amplification products but        are not required.

-   3. Analyze the PCR product by agarose gel electrophoresis and    quantify the DNA yield using the PicoGreen dsDNA kit.    -   The typical yield for a 120 bp amplicon is in the order of ˜15        ng/μl (˜200 nM). Excess primer from the pre-amplification        competes with the primer on the beads and decreases the amount        of PCR product bound to beads during the Emulsion PCR process.        If the concentration of amplicon is less than 4 nM, then purify        the PCR product with a QIAquick PCR purification kit to remove        the PCR primers.    -   TROUBLESHOOTING    -   PAUSE POINT DNA can be stored at −20° C.    -   CRITICAL STEP

Binding of Primers to Beads

-   4. In a 1.5 ml microcentrifuge tube, wash 100 μl (7-12×10⁸)    streptavidin-coated magnetic beads twice with 100 μl TK buffer.    After each wash, place the tube on a magnet for 1 min to concentrate    the beads and remove the supernatant with a pipette.    -   This will result in enough beads to perform 18 emulsion PCRs.-   5. Resuspend the beads in 100 μl Binding buffer and add 10 μl Primer    4 (100 μM, in TE buffer). Vortex immediately.-   6. Incubate the bead suspension at 15-25° C. for 30 min. Every 10    minutes or so, mix the beads by briefly vortexing the tube.-   7. Separate the beads, now coated with primers, with the magnet.    Remove the supernatant and wash the beads 3 times with TK buffer as    described above.-   8. Resuspend the beads in 100 μl TK buffer.    -   PAUSE POINT Beads can be stored at 4° C. for at least 3 months.    -   CRITICAL STEP

Emulsion PCR

-   9. Prepare emulsifier/oil mix within one or two days of use.    -   A precipitate in the Tegosoft DEC oil is occasionally observed        in the bottom of the bottle; do not include this precipitate        when preparing the mix.-   10. Dilute the template DNA with TE to ˜20 pM immediately prior to    use.    -   DNA at low concentrations can stick to tubes during storage.-   11. Set up a 150 μl amplification reaction by mixing the following:

Primer 5 (2.5 μM) 3 μl Primer 6 (400 μM) 3 μl Template DNA (~20 pM) 10μl  Beads 6 μl dNTPs mix 3 μl 10x PCR buffer 15 μl  Platinum Taq DNApolymerase (5 U/μl) 9 μl Water 101 μl 

-   -   CRITICAL STEP

-   12. Add, in order, one steel bead, 600 μl oil/emulsifier mix, and    150 μl PCR reaction mix to one well of a 96-well storage plate. Seal    plate with adhesive film.    -   The adhesive film will not seal properly, if oil is present on        the rims of the wells. Turn the plate upside down to make sure        the steel bead moves freely in the well.    -   CRITICAL STEP

-   13. Assemble a TissueLyser adaptor set by sandwiching the 96-well    storage plate containing the emulsion PCR mix between the top and    bottom adapter plates each fitted with a compression pad facing the    96-well storage plate. Place the assembly into the TissueLyser    holder, and close the handles tightly. Mix for 10 s at 15 Hz and for    7 s at 17 Hz.    -   Balance TissueLyser with a second adaptor set of the same        weight.    -   CRITICAL STEP

-   14. Disassemble the adaptor set and centrifuge the plate for 10 sec    at ˜3 g to get the liquid to the bottom

-   15. Assess the quality of the emulsions at 400× magnification with    an inverted microscope.    -   Take a pipette tip, dip it into the emulsion, and streak it over        the bottom of a 48-well cell culture plate. Do not use        coverslips, as these can alter the quality of the emulsion.    -   Examine sample immediately as the aqueous compartment evaporates        quickly. FIG. 2 shows a photograph of emulsions prepared by this        process.    -   TROUBLESHOOTING

-   16. Aliquot 80 μl of the emulsion into eight wells of a 96-well PCR    plate.    -   Pipette emulsions slowly to avoid shear force. Centrifuge the        plate for 10 sec at ˜3 g to get the liquid to the bottom.

-   17. Temperature cycle the emulsions according to the following    program:

Cycle number Denaturation Annealing Polymerization 1  2 min at 94° C.2-4 15 s at 98° C. 45 s at 64 s 75 s at 72° C. 5-7 15 s at 98° C. 45 sat 61 s 75 s at 72° C.  8-10 15 s at 98° C. 45 s at 58 s 75 s at 72° C.11-60 15 s at 98° C. 45 s at 57 s 75 s at 72° C.

-   -   PAUSE POINT Emulsions can be stored at 4° C.

Breaking Emulsions

-   18. To each 80 μl emulsion, add 150 μl Breaking buffer and pipette    up and down 3 times to mix.-   19. Seal the PCR plate, place it into an empty 96-well storage    plate, and assemble between two TissueLyser adaptor plates as    described above (Step 13). Place in TissueLyser and mix for 30 s at    20 Hz.-   20. Remove PCR plate from the TissueLyser and centrifuge for 2 min    at 3200 g.-   21. Remove the top oil layer with a 20 μl pipette tip attached to a    vacuum manifold.-   22. Add 150 μl Breaking buffer, seal the plate, and centrifuge again    for 2 min at 3200 g.-   23. Place the plate in a 96-well magnetic separator for 1 min and    completely remove the liquid with a pipette.-   24. Remove the plate from the magnet, resuspend the beads in 100 μl    TK buffer, and pool the beads from the eight wells into a 1.5 ml    tube.-   25. Place the tube on the magnet to concentrate the beads for 1 min    and carefully remove the supernatent with a pipette.    -   TROUBLESHOOTING-   26. Resuspend beads in 500 μl of 0.1 M NaOH and incubate for 2 min.    Place the tube in magnetic separator for 1 min and carefully remove    supernatant.    -   This removes the non-biotinylated DNA strand from the beads.-   27. Resuspend the beads in 100 μl TK buffer.    -   The recovery of beads can be assessed by measuring absorption at        600 nm. The Nanodrop spectrophotometer is convenient for this        purpose as it only requires 2 μl bead suspension. An aliquot of        the beads coated with Primer 5 can be used as a fiducial. The        typical recovery with the procedure described above is 50-70%.    -   TROUBLESHOOTING    -   PAUSE POINT Beads can be stored at 4° C.

Detection of DNA on the Beads

-   28. Set up the oligohybridization in a 96-well PCR plate by mixing    the following:

Primer 3 (1 μM) 10 μl Beads 20 μl 5x hybridization buffer 20 μl Water 60μl

-   -   The number of beads to be used depends on the nature of the        experiment. Ten million beads provide a great enough mass to be        seen during magnetic collection and facilitate recovery. The        recovery can be assessed by measuring absorption at 600 nm as        described above.

-   29. Incubate at 50° C. for 15 min in a thermal cycler.

-   30. Place the plate on a 96-well magnetic separator for 1 min to    concentrate the beads and remove 80 μl of the supernatant with a    pipette.

-   31. Wash beads twice with 80 μl TK buffer.    -   PAUSE POINT Beads can be stored at 4° C.

Analysis of Bead Populations

-   32. Use flow cytometry to determine the relative fluorescence    intensity of the primers hybridized to the DNA on the beads.    -   We have successfully used the BD Bioscience FACScan, LSR I & II,        FACSCalibur, and FACSAria. Alternatively, fluorescence        microscopy can provide a rapid qualitative analysis of the beads        generated.    -   CRITICAL STEP*-   33. Empirically, establish the amplifier gain (voltages) for the    detection of the forward scatter (FCS), side scatter (SSC), and    fluorescence signal.    -   FIG. 3 illustrates typical results obtained.    -   TROUBLESHOOTING**    -   CRITICAL STEP*

Critical Steps

Critical Step 3

The amount of DNA used in the emulsion PCR can be varied over arelatively wide range. Optimally, 20%+/−15% of the beads should containPCR products. Too little template results in too few positive beads,compromising the sensitivity of analysis. Too much template results intoo many compartments containing multiple templates, making it difficultto accurately quantify the fraction of initial templates containing thesequence of interest.

Critical Step 8 Many beads in addition to the 1.05 micron MyOne beadscan be used for this procedure. MyOne beads are uniform, which isespecially advantageous for flow cytometry. Other magnetic beads (suchas Sera-Mag particles from Seradyn) have more surface streptavidinmolecules than MyOne and can be used when surface density is moreimportant than uniformity. Larger uniform beads (such as DynabeadsM-280) have even more surface streptavidin molecules, but are much moreexpensive (per bead) and the emulsion formulation must be altered tomake the them efficient supports. Finally, non-magnetic beads can beused, though these are more difficult to handle because centrifugationrather than magnets must be used to manipulate them.

Critical Step 11 The efficiency of amplification on solid supports inemulsions decreases with increasing amplicon length³. The preferredamplicon length (including primers) is 70 to 110 bp. Amplicons of 200 bpyield ˜30% of the product of those containing 100 bp on beads. Wegenerally use a universal primer (Primer 5 in FIG. 1) as the reverseprimer. However, one can also use a nested reverse primer that resultsin an amplicon shorter than the product of the pre-amplification step toreduce nonspecific amplification on the microspheres or to decrease thesize of the bead-bound PCR product and thereby increase yield. Finally,the concentration and type of polymerase has been extensively optimizedin the protocol described here. In general, higher polymeraseconcentrations result in higher yields of PCR products bound to beads.Another way to increase the amount of PCR product bound to the beads isthrough rolling circle polymerization².

Critical Step 12 If a TissueLyser is not available, emulsions can alsobe generated using a stir-bar or a homogenizer^(1,4). Though theemulsions are not as uniform or as easily controlled as those made withthe TissueLyser, they are adequate for many applications of BEAMing,especially when only a small number of samples is required. One simpleway to prepare such emulsions is by mixing 240 μl PCR reaction with 960μl 7% Abil EM90 in mineral oil (Sigma) in a 2 ml cryogenic vial(Corning, 430661) for 10 sec with a vortexer and for 50 sec with ahomogenizer (IKA, T25 basic, 2953000) equipped with a disposablehomogenizer tip (Omni Intl., 30750) at minimum speed⁴.

Critical Step 13 When using the 96-well storage plate with theTissueLyser adaptor plates, samples closer to the body of the instrument(rows G and H) vibrate more slowly than samples in rows A and B. Toprevent variation in emulsion quality, we recommend using only rows Aand B if <24 samples are being prepared. When using the entire 96-wellplate, rotate the to 96-well plate half way through the mixing process.

Critical Step 32 In the single tube operation mode of the FACSCaliburand LSR I & II instruments, the droplet containment module (DCM) sleeveabove the sample injection tube (SIT) should be removed to prevent the 1micron magnetic beads from getting trapped between the sleeves. Thesleeve can be replaced by a shorter modified metal sleeve protector. Wealso recommend using high quality sheath fluid.

Critical step 33 The forward scatter resolution of instruments using atraditional photodiode detector should be sufficiently sensitive todetect single 1 micron particles. However, if not properly aligned thebeads will be difficult to separate from background. A forward scatterphotomultiplier tube (PMT) detection system increases sensitivity downto a resolution of 0.2 μm and is recommended when using beads of 1micron in diameter.

Troubleshooting Table

Problem Step 3 Desired Band is not the Dominant Product of thePre-Amplification. Solution

Use a higher annealing temperature (60 to 65° C.) and vary the MgCl₂concentration (1.5 mM to 2.5 mM). GC-rich templates should be amplifiedin Phusion GC buffer and 3-6% DMSO. Check that the sequence is notrepeated in the template genome. Check for homodimer and heterodimerformation with an oligo analyzer program (e.g. OligonucleotideProperties Calculator;http://www.basic.northwesterwedu/biotools/oligocalc.html). It is worthtrying several primer pairs if amplification is a problem.

Problem Step 15 Aqueous Droplets are Too Small or Too Big. Solution

If the aqueous compartments of an emulsion are too small there will belittle or no amplification. If the compartments are too large, thefraction of compartments with only a single template will be too low toprovide a statistically significant result. The droplet sizes can beoptimized varying the mixing time or vibration speed by increments of 1s or 1 Hz, respectively.

Problem Step 25 Beads Form Visible Aggregates. Solution

We have occasionally observed aggregates of magnetic beads at varioussteps after breaking the emulsions. This occurs more frequently withlarger amplicons (>200 bp). The factors that might be responsible arethe time a sample is placed in the magnetic field, the saltconcentration and the temperature of the buffers. In order to minimizethe likelihood of aggregate formation use only buffers equilibrated atroom temperature. The samples should not be left on the magnet for morethan 2 min (especially after the liquid has been removed). The saltconcentration can be increased. Once aggregates have formed they arehard to disperse. In some cases they can be dispersed by pipetting,vortexing, or sonicating (Bioruptor from Diagenode). Heating also canhelp reverse aggregation in some cases.

Problem Step 27 Low Recovery of Beads after Emulsion PCR.

Solution

Low recoveries can result from incomplete demulsification or inefficientmagnetic separation. To prevent incomplete demulsification increase themixing time and speed. To prevent loosing beads during the magnetseparation never remove all of the supernatant, do not touch the beadpellet, and use the same brand of tubes, plates and magnets as suggestedabove.

Problem Step 33 Poor Signal to Noise. Solution

This is usually due to a low efficiency of on-bead amplification but canbe due to substandard hybridization conditions or to a poor probe (e.g.secondary structure preventing hybridization). Purification ofhybridization probes by HPLC or gel electrophoresis is recommended.Check the hybridization conditions by hybridizing a primer to the beadsthat is complementary to Tag1. The signal obtained represents themaximal signal possible, as every PCR product on the bead has thissequence at its 5′ end. The signal from these beads should be equivalentto those achieved by binding a 5′-biotinylated oligonucleotide that hasa similar fluorescent group on the 3′ end. The signal from Primer 3should be within 10-fold of the signal obtained with Tag1, meaning thatat least 10% of the bead-bound primers were extended during the PCR. Lowamounts of DNA on the beads can be caused by a small size of the aqueouscompartments (see Problem Step 15), or poor reaction conditions. Notethat extensive optimization has been performed on every aspect of theprotocol described herein and deviations from these conditions should beundertaken with caution.

REFERENCES

The disclosure of each reference cited is expressly incorporated herein.

-   1. Dressman, D., Yan, H., Traverso, G., Kinzler, K. W. &    Vogelstein, B. Transforming single DNA molecules into fluorescent    magnetic particles for detection and enumeration of genetic    variations. Proc Natl Acad Sci USA 100, 8817-8822 (2003).-   2. Li, M., Diehl, F., Dressman, D., Vogelstein, B. & Kinzler, K. W.    BEAMing up for detection and quantification of rare sequence    variants. Nat Methods 3, 95-97 (2006).-   3. Shendure, J. et al. Accurate multiplex polony sequencing of an    evolved bacterial genome. Science 309, 1728-1732 (2005).-   4. Diehl, F. et al. Detection and quantification of mutations in the    plasma of patients with colorectal tumors. Proc Natl Acad Sci USA    (2005).-   5. Utada, A. S. et al. Monodisperse double emulsions generated from    a microcapillary device. Science 308, 537-541 (2005).-   6. Nagai, H., Murakami, Y., Morita, Y., Yokoyama, K. & Tamiya, E.    Development of a microchamber array for picoliter PCR. Anal Chem 73,    1043-1047 (2001).-   7. Mastrobattista, E. et al. High-throughput screening of enzyme    libraries: in vitro evolution of a beta-galactosidase by    fluorescence-activated sorting of double emulsions. Chem Biol 12,    1291-1300 (2005).-   8. Margulies, M. et al. Genome sequencing in microfabricated    high-density picolitre reactors. Nature 437, 376-380 (2005).-   9. Kojima, T. et al. PCR amplification from single DNA molecules on    magnetic beads in emulsion: application for high-throughput    screening of transcription factor targets. Nucleic Acids Res 33,    e150 (2005).

1-12. (canceled)
 13. A liquid composition comprising a plurality of microemulsions forming aqueous compartments wherein at least a portion of said aqueous compartments comprise: a bead; a polynucleotide template; and oligonucleotide primers for amplifying said template; wherein at least a portion of the oligonucleotide primers is bound to the bead, wherein said microemulsions comprise an oil phase and an aqueous phase, wherein the aqueous phase comprises from 10-30% (v/v) of the microemulsions and the oil phase comprises from 70-90% (v/v) of the microemulsions; wherein the oil phase comprises one or more low viscosity hydrocarbons with a viscosity less than 20 mPas at 25° C. in an amount from 60-85% (v/v) of the oil phase, one or more high viscosity hydrocarbons having a viscosity of greater than 20 mPas at 25° C. in an amount from 10-30% (v/v), and an emulsifier in an amount from 5-10% (v/v).
 14. The liquid composition of claim 13 wherein the microemulsions comprise the high viscosity hydrocarbons in an amount from 15-25% (v/v) of the oil phase.
 15. The liquid composition of claim 13 wherein the microemulsions comprise the high viscosity hydrocarbons in an amount from 17-23% (v/v) of the oil phase.
 16. The liquid composition of claim 13 wherein a plurality of separate microemulsion populations are formed simultaneously in a multi-well plate using a tissue mixer mill disrupter and a metal ball in each well.
 17. The liquid composition of claim 13 wherein the low viscosity hydrocarbons comprise an oxygen moiety selected from the group consisting of: a hydroxyl, an ester, an ether, and a carboxylic acid.
 18. The liquid composition of claim 13 wherein the high viscosity hydrocarbons have a viscosity of 20-30 mPas at 25° C.
 19. The liquid composition of claim 13 wherein the high viscosity hydrocarbons have a viscosity of 20-25 mPas at 25° C.
 20. The liquid composition of claim 13 wherein the high viscosity hydrocarbons have a viscosity of 22-26 mPas at 25° C.
 21. The liquid composition of claim 13 wherein the low viscosity hydrocarbons are selected from the group consisting of: 4-glyceryl isostearate, ethylene glycol, propylene glycol, cetyl propylene glycol, hexyl laurate, diethyl hexylcarbonate, and mixtures thereof.
 22. The liquid composition of claim 13 wherein the low viscosity hydrocarbons have a viscosity of less than 15 mPas at 25° C.
 23. The liquid composition of claim 13 wherein the low viscosity hydrocarbons have a viscosity of less than 10 mPas at 25° C.
 24. The liquid composition of claim 13 wherein the low viscosity hydrocarbons have a viscosity of less than 5 mPas at 25° C. 25-38. (canceled) 